1. Field of the Invention
The present invention generally relates to chronic lymphocytic leukemia (CLL) detection, prevention and therapy and more specifically to antibodies directed against antigens specific for CLL, gene therapy for CLL, and inhibition of ROR1 expression and activity.
2. Background Information
Patients with cancer can develop immune responses against tumor-associated antigens (TAAs) and potentially reject autologous tumor. Many such TAAs represent developmental or differentiation antigens that have restricted expression. Conceivably, chronic lymphocytic leukemia (CLL) also might have such leukemia-associated antigens (LAA). Microarray analyses revealed that there are genes expressed by CLL cells that are not expressed in other lymphoid tissues. Coupled with the observation that CLL cells also express a highly restricted immunoglobulin repertoire, cells likely express distinctive antigens that also could be targeted for immunotherapy. However, CLL patients typically develop hypogammmaglobulinemia and worsening immune deficiency, which impairs their immune response to vaccines. Implicated in the abnormal immune function are immune-suppressive factors and an acquired functional deficiency of CD154. Furthermore, CLL cells are particularly poor at antigen presentation, which appears in part secondary to inadequate leukemia-cell expression of immune co-stimulatory/adhesion molecules.
Activation of CLL cells via CD40-ligation can reverse its immune-suppressive phenotype. Furthermore, CLL cells transduced with an adenovirus encoding the ligand for CD40 (Ad-CD154) can function as more effective antigen-presenting cells (APCs). In addition, they can effect ligation of CD40 on bystander leukemia B cells and stimulate autologous leukemia-reactive T cells both in vitro and in vivo.
Tyrosine kinases are important mediators of the signaling cascade, determining key roles in diverse biological processes like growth, differentiation, metabolism and apoptosis in response to external and internal stimuli. Studies have implicated the role of tyrosine kinases in the pathophysiology of cancer. Schlessinger J. (2000) Cell, 103:211-225; and Robinson et al. (2000) Oncogene, 19:5548-5557. MacKeigan and colleagues used a large-scale RNAi approach to identify kinases that might regulate survival and apoptosis of a human tumor cell line (HeLa), RNAi to ROR1 was found as one of the most potent in inducing apoptosis among the set of RNAi targeting each of 73 different kinase-encoding genes. MacKeigan et al. (2005) Nat Cell Biol., 7:591-600. However, these investigators did not examine the expression or function of ROR1 protein in these cells.
ROR1 is a membrane-receptor with an intracellular kinase-like domain and extracellular Frizzled-like cysteine-rich domain, which is common to receptors of members of the Wnt-family. ROR1 is member of the ROR family that is evolutionarily conserved among Caenorhavditis elegans, Drosophila, mice and humans. Wilson C, Goberdhan D C, Steller H. Dror, a potential neurotrophic receptor gene, encodes a Drosophila homolog of the vertebrate Ror family of Trk-related receptor tyrosine kinases. Proc Natl Acad Sci USA. 1993; 90:7109-7113; Oishi et al. (1997) J Biol. Chem., 272:11916-11923; Masiakowski et al. (1992) J Biol. Chem., 267:26181-26190; Forrester et al. (2002) Cell Mol Life Sci., 59:83-96; and Oishi et al. (1999) Genes Cells, 4:41-56. In rodents, ROR1 is expressed primarily in developing cephalic neural crest in the dorsal part of the diencephalons and mid-hind brain boundary during embryogenesis. In most species examined, expression of ROR1 apparently attenuates during embryonic development, becoming negligible at term. ROR1 mRNA was reported to express in infant brain, renal cancer and colon cancer. In a recent study, it was found that ROR1, at both mRNA and protein level, was highly expressed in CLL B cells but not normal B cells. Moreover, it was found that ROR1 is a receptor for Wnt5a, which could induce activation of NF-κB when co-expressed with ROR1 in HEK293 cells and enhance survival of CLL cells in vitro. This indicates that ROR1 is a CLL survival-signaling receptor for Wnt5a, however direct evidence of ROR1 effect on CLL progression is required. Another study found that ROR1 was expressed in acute lymphocytic leukemia (ALL) as well. Shabani et al. (2007) Tumour Biol., 28:318-326; and Baskar et al. (2008) Clin Cancer Res., 14:396-404. However, ROR1 expression on development and progression of other cancers has not yet been extensively explored, and therapeutic control of ROR1 expression is necessary.
As discussed above, ROR1 is an embryonic protein that is expressed uniquely on certain cancer cells, including in CLL, small lymphocytic lymphoma, marginal cell B-Cell lymphoma, Burkett's Lymphoma, and other cancers (e.g., breast cancers), but not on normal adult tissues and cells. Anti-ROR1 antibodies raised against ROR1 peptide are commercially available, but monoclonal anti-ROR1 antibodies that react with the native ROR1 protein have not been made or isolated. In addition, no anti-ROR1 antibodies capable of detecting cell-surface expression of ROR1 for flow cytometric analysis have been made or isolated. The antibody that can react with native ROR1 protein are also necessary.